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Where Iron and Immunity Intersect

Charles Fergus

May 1, 2003

Where Iron and Immunity Intersect

Visualize the villi of the small intestine as a field of corncob-shaped protrusions, a complexity of tissue whose absorptive surface area covers thousands of square feet. A single villus measures 0.02 to 0.04 inch in length, or about as long as two or three playing cards are thick. It is coated with epithelial cells: like kernels on the cob. An epithelial cell, or enterocyte, lives for about three days. It originates in the crypt, a low-lying portion of the villus; pushed by cell division, it migrates slowly upward. When it reaches the villus tip, the exhausted cell sloughs off and disintegrates, spilling its contents—including iron gleaned from food—back into the intestine, ultimately to be excreted.

James Collins

It is the middle part of the enterocyte's journey that fascinates Michael Chorney: the time during which the cell takes up iron, an element necessary for life but dangerous when accumulated in excess. Chorney, an immunologist and geneticist at Penn State's Hershey Medical Center, is the lead investigator in a five-year program project grant, Molecular Dynamics of Iron Regulation and Function, awarded by the National Institutes of Health. The grant, funded from 1999 through 2003, supports the work of three laboratories—Chorney's and two others, also at Hershey—examining different aspects of iron metabolism.

Chorney comes at the problem through the immune system. Twenty-five years ago, he was at Sloan-Kettering Cancer Center in New York, working toward his Ph.D. and collaborating with a group of researchers conducting molecular mapping of the major histocompatibility complex, or MHC, in laboratory mice. The MHC includes several hundred genes, many of which help direct the body's immune response by regulating T lymphocytes, white blood cells that destroy pathogens and cause the body to reject skin grafts and transplanted organs.

Chorney's curiosity was piqued by another, nearby gene, an outlier from the MHC cluster but on the same chromosome and having the same general sequence as many MHC genes. Through experiments using mice, Chorney learned that this gene also communicates with T lymphocytes—not to spur the immune response, but to help the body regulate the uptake and storage of iron. Chorney predicted that a comparable gene would be found in the human genome, and that it would have a similar function.

Today, the gene has been located on the short arm of human chromosome 6. It's been given a name: HFE. It is sometimes referred to as the hemochromatosis disease gene, because a mutation in its genetic code can lead to hemochromatosis, an insidious disease also known as iron overloading.

Iron, nearly ubiquitous in nature, is present in every cell. It is a component of hemoglobin in red blood cells, binding the oxygen that the blood circulates throughout the body. We need iron for strength and vigor, and the element plays a key role in DNA and enzyme synthesis and other basic life processes.

Michael Chorney

These liver cells have loaded up on iron — a substance necessary to the body but harmful when absorbed in excess.

Under normal conditions, iron is tied up by small molecules, known as chelators, in the cytoplasm of individual cells. When iron is abundant—supplied by a diet rich in iron, for instance—the body stores some of it in the enterocytes coating the villi of the small intestine. Should the body need an iron boost—after blood loss, for example—the enterocytes release iron to the blood stream. If iron stores are adequate and there is no immediate need for the element, the enterocytes hang on to their iron; when they die and slough off at the villus tip through the process known as apoptosis, or programmed cell death, the body rids itself of the excess iron.

Most of us are familiar with anemia, a common disorder in which a dearth of iron leads to pallid skin, dizziness, and shortness of breath. Fewer know of hemo-chromatosis, a heritable disease afflicting between one and two million people in the United States and millions more in northern Europe, the British Isles, and Australia. Although it's not as widespread as anemia, hemochromatosis occurs much more frequently than cystic fibrosis, often touted as the most common autosomal recessive disorder in humans.

Iron flow into the body is like waves on the shore—it's a constant, says Chorney. Healthy people dump their excess iron back into the intestine. But in persons with hemochromatosis, iron absorption is enhanced. The body collects iron beyond what is needed, and fails to get rid of it. Sufferers can accumulate as many as 20 to 40 grams of iron, compared to 3 to 4 grams in normal adults. When that happens, the skin may take on a bronze tinge. Deeper in the body, iron builds up in joints, causing fatigue and pain. It lodges in pancreatic cells, cardiac cells, and liver cells, often leading to diabetes, heart disease, cirrhosis, and liver cancer; doctors have characterized hemochromatosis as a rusting away of the body's tissues.

Women with hemochromatosis tend to develop symptoms later than men, because women shed iron through blood loss during menstruation and childbirth. In men, problems usually begin showing up around age 40 or soon after. A longterm course of phlebotomy—the regular drawing off of blood, also known as bloodletting—can bring relief; a low-iron diet also helps. The effects of hemochromatosis can be reversed if the disease is diagnosed early enough. But by the time symptoms appear, serious damage often has already occurred.

In 1994, Chorney began studying blood samples from 200 hemochromatosis sufferers, many of them undergoing treatment at the Medical Center. Using a technique called linkage disequilibrium mapping, he searched for shared genetic markers among the patients. The closer we got to the disease gene, he says, the more similarities began showing up among the genetic markers. In one patient, Chorney found that a piece of chromosome 6 was actually cut out and flipped around backwards—a one-in-a-million shot, says Chorney, that helped us home in on the gene.

At the time, Chorney was communicating closely with fellow researchers from around ten laboratories worldwide hunting for the hemochromatosis gene. Chorney's gene-mapping results, together with data uncovered by other scientists, helped a California firm, Mercator Genetics, identify and clone the gene; Chorney ultimately collaborated with Mercator to verify that the gene they'd cloned was the cause of hereditary iron overloading.

Even though hemochromatosis patients have elevated iron levels, says Chorney, their enterocytes and macrophages—key cells in the iron intake and redistribution pathway—remain iron-poor. To understand why this is so, and to develop possible strategies for treating and preventing hemochromatosis, we need to study the intake and transfer of iron on both the cellular and molecular levels.

Michael Chorney

A microscopic thin-section from the intestine showing the villi, structures that absorb iron during digestion.

Chorney and his research team—consisting of postdoctoral scholars, dual M.D./ Ph.D. candidates, graduate students, and scientists from other institutions—have scrutinized the region in the small intestine between the iron-absorbing enterocytes and the nearby capillaries that ferry iron to the rest of the body. This interstitial space contains a fluid swimming with T lymphocytes and other immune-system cells.

In a parallel research project focusing on evolutionary aspects of the major histo-compatibility complex, Chorney and colleague Austin Hughes at the University of South Carolina noted similarities between the HFE gene and a related gene known as MIC. MIC stimulates immune cells to produce certain proteins in response to various forms of physical stress; one of those proteins, tumor necrosis factor, attaches to tumor cells so they can be recognized and attacked by lymphocytes. Chorney wondered if the HFE gene, functioning properly, might let iron-rich enterocytes signal nearby T lymphocytes that iron-loading—itself a form of stress—was taking place. Then the T lymphocytes could respond and control the release of iron from the enterocytes.

Chorney discovered that when laboratory mice were fed iron-laden chow, their lymphocytes began manufacturing tumor necrosis factor. As part of the immune response, tumor necrosis factor promotes inflammation: redness, swelling, heat, and pain, conditions reflecting an increased blood flow, which spurs a buildup of lymphocytes able to remove tumor cells or kill invading microbes. Is tumor necrosis factor, Chorney asked, the direct response to the HFE flag signaling the need to keep iron locked up in the enterocytes?

Chorney ran a second experiment using a line of mice bred to model hemochromatosis patients. When fed the same iron-rich diet, the mice failed to produce tumor necrosis factor. Using iron-staining techniques and atomic absorption spectroscopy, the researchers found elevated levels of iron in the animals' livers and no evidence of iron deposition in their enterocytes, indicating, says Chorney, that iron was flowing unchecked into the blood and on into the liver.

In The Enigmatic Role of the Hemochro-matosis Protein (HFE) in Iron Absorption, a paper published in the March 2003 Trends in Molecular Medicine, Chorney proposes a piggyback-sensor model explaining how iron uptake may go awry in hemochromatosis. The mechanism involves transferrin, a blood plasma protein that transports iron; and transferrin receptor, a protein on the surface of many cells that mediates the uptake of transferrin and the release of its iron atoms, two of which are usually bound by each transferrin molecule.

In the enterocyte, suggests Chorney, transferrin receptor binds to the HFE protein, abolishing its communications function by piggybacking it into acid-rich endosomes (some 75 percent of a cell's transferrin receptor collects in the endosomes, compartments where iron atoms are pried loose from transferrin proteins). With the HFE protein out of sight, says Chorney, neighboring T lymphocytes consider that all is well with iron levels in the enterocyte: That's the normal state in a person who does not have hemochromatosis and is eating a normal iron diet.

In someone whose HFE carries the disease-causing mutation, even as an enterocyte's iron content increases, the cell continues to believe the body needs iron. And no flag is raised telling nearby lymphocytes that iron levels are high. The lymphocytes fail to produce tumor necrosis factor, which, under normal conditions, would enhance lymphocyte activity while forcing the enterocytes to hold on to their iron. Iron continues to move unchecked from the enterocytes into the blood stream.

Says Chorney, The general redundancy of immune system elements and pathways probably means that the effects of mutant HFE molecules are subtle, part of a larger overall picture.

Here's where it gets really interesting to me, Chorney muses. Suppose the HFE gene is a remnant of an older immune response, something that evolved long ago in vertebrates. Perhaps its current role in fine-tuning iron levels in the intestine evolved from its former role in fighting infection. What if it does that by using the T lymphocytes originally intended to attack microbes, creating a local, fragmentary inflammation response? That would explain why patients with chronic infections often develop anemia.

Michael Chorney

A single villus measures 0.02 to 0.04 inch in length, or about as long as two or three playing cards are thick.

Chorney points at the immune system cells known as macrophages: Think of them as professional eaters. Macrophages are involved in the process in the bone marrow whereby old red blood cells are broken down and the iron in their hemoglobin is recycled. Says Chorney, The high-iron status of the macrophages may cause a tumor necrosis response much like the one that occurs in the intestine.

During infection by mycobacteria—the bugs that cause pneumonia—macro-phages seemingly use iron to protect the body. The macrophages engulf the bacteria. Then they may pump iron into endosomelike chambers holding the bacteria, killing them directly; or they may withhold iron from the bacteria, keeping them from reproducing and preventing the infection from spreading. This further suggests that the use of iron may be one of the body's oldest immune defensive strategies.

It's no coincidence that the hemochromatosis gene is related to the major histo-compatibility complex genes that regulate immunity. It makes sense that the expressionof HFE in the intestine and the macrophages translates into a type of limited inflammation. But instead of fighting infection, perhaps HFE-induced inflammation prevents iron's unrestricted passage from the enterocytes to the blood.

Chorney admits that his hypotheses are highly novel in the hemochromatosis and iron field. He adds, Iron is something of a mystery. For instance, we don't know precisely how iron moves from the enterocytes to the capillaries in the intestine. We do know that its aberrant flow and storage have an impact on many diseases.

Consider brain disease and liver cancer. At Hershey, funded by the same program project grant for which Chorney is the lead investigator, neuroscientist James Connor is studying iron uptake and storage in the brain, particularly the proteins that regulate the element in individual cells. Connor's research has contributed to a greater understanding of the molecular aspects of cell damage leading to Alzheimer's and Parkinson's diseases. Iron in the diseased brain may accumulate from the activation and iron unloading of resident macrophages. Or iron's transport across the blood-brain barrier may become faulty, stimulating macrophages that turn against the body and destroy brain tissue.

Microbiologist Harriet Isom is developing a cell culture model—an in vitro model to be used in the lab for the controlled study of iron regulation in the liver. She is focusing on oxidative cell damage caused by too much iron in hepatocytes (liver cells), thought to be a major factor in the development and spread of liver cancer. In the liver, iron induces toxic radical compounds that may attack and destroy other organic compounds, including DNA, leading to a malignancy. The fact that many hemochro-matosis patients die of liver cancer offers support for this possibility, notes Chorney.

Chorney and colleagues Wen-Jie Zhang and Walter Koltun in Hershey's department of surgery have been studying inflammatory bowel diseases, including ulcerative colitis and Crohn's disease. Inflammatory bowl diseases are widespread in western society; some medical researchers blame them on our iron-rich diet. The afflictions arise when tissues lining the gastrointestinal system are attacked by the body's immune system.

Says Chorney, It's possible that the control of iron flow by the T lymphocytes causes an initial inflammation that later escalates into a full-blown inflammatory attack, following entry into the intestine of outside pathogens. He notes that these two devastating illnesses are routinely treated using antibodies that neutralize the action of tumor necrosis factor. Maybe someday we'll conclude that hemochromatosis and the misregulation of iron are what cause those diseases in the first place.

Michael J. Chorney, Ph.D., is professor of microbiology and immunology and pediatrics, H107, Hershey Medical Center, Hershey PA 17033; 717-531-4604; mjc18@psu.edu. The five-year program project grant is from the National Institute of Diabetes and Digestive and Kidney Diseases in the National Institutes of Health. James R. Connor, Ph.D., is professor of neuroscience and anatomy and interim chair of the department of neuroscience and anatomy, H109, Hershey Medical Center, Hershey PA 17033; 717-531-6408; jrc3@psu.edu. Harriet C. Isom, Ph.D., is distinguished professor of microbiology and immunology, H107, Hershey Medical Center, Hershey PA 17033; 717-531-8609; hci1@psu.edu. Isom is also assistant dean for the M.D./Ph.D. program at Hershey.